Optimizing viral protein yield of influenza virus strain A/Vietnam/1203/2004 by modification of the neuraminidase gene

Abstract

The preparation of high-yield prepandemic influenza virus H5N1 strains has presented a challenge to both researchers and vaccine manufacturers. The reasons for the relatively low yield of the H5N1 strains are not fully understood, but it might be partially dependent on the interactions between the hemagglutinin (HA) or neuraminidase (NA) surface glycoprotein and other influenza virus proteins. In this study, we have constructed chimeras between the A/Puerto Rico/8/34 (PR8) NA gene and the A/Vietnam/1203/2004 (VN1203) NA gene that have resulted in an increase in the virus yield of the reassortant viruses without a significant loss of NA activity. By combining the amino terminus of NA from the PR8 strain with the carboxy terminus of NA from VN1203, the surface epitopes unique to the H5N1 VN1203 NA glycoprotein are maintained. This reassortant virus had a higher titer and total protein yield in eggs, grew to a higher titer, produced large plaques on MDCK cells, and retained NA activity. This work describes a novel recombinant technique designed to increase the yields of vaccine candidates for the production of pandemic influenza virus vaccines. The relationship between the infectivity and protein yield of the reassortants also is discussed.

FIG. 1.

Neuraminidase alignment and chimeras. (A) The alignment of the amino terminus of the NA gene segments from A/Vietnam/1203/2004 (top line) and A/Puerto Rico/8/34 (bottom line) is shown. The Clustal W method was used for this alignment, and identical amino acids are shown with taller bars, while changes are shown with shorter bars. The first six amino acids comprise the only cytoplasmic portion of the protein (1). The NA transmembrane region is boxed, and the stalk region is underlined. Deletions are indicated by a dash instead of an amino acid letter. (B) The first two constructs shown in this cartoon are PR8 (striped) and VN1203 (solid), respectively. Chimeras between the two NA genes are indicated by the insertion of the striped PR8 portion into the solid VN1203 NA gene, and the amino acid numbers indicate the length of the final constructs.

FIG. 2.

Plaque morphology of the reassortant viruses on MDCK cell monolayers. Cell monolayers were fixed with methanol at 72 h postinfection and were stained with 1% crystal violet. The plates were dried, and then plaques were counted and photographed for an analysis of the various plaque sizes.

FIG. 3.

Replication kinetics. Single-step growth curves of reverse genetics-generated viruses in MDCK cells. Cultures in 12-well dishes were infected at a multiplicity of infection of 3 PFU/cell, and samples were taken from the infected cell monolayer at the indicated time points. The titers of the samples on MDCK cells were determined. These growth curves are averages from three independent experiments, and standard errors are indicated. N-term, N terminus; aa, amino acid.

FIG. 4.

Total protein. The viruses were propagated in the allantoic cavities of 9-day-old embryonated eggs by the inoculation of 0.2 ml of diluted virus stocks containing ∼10⁴ EID₅₀ or 0.1 HA units/ml at 33°C for 2 days. Virus in allantoic fluid was concentrated and purified by banding in 15 to 60% sucrose gradients (14). Total protein yield was measured from virions purified from 60 ml of allantoic fluid. Viruses that contained the HA gene from PR8 are the striped bars on the left, while those with the VN1203 HA gene are represented by the solid bars on the right. The JV15 (wild-type) virus that contains HA and NA from PR8 was set at 100% for protein yield, and the remainder of the viruses are compared to this. This graph shows the averages from two independent experiments. JV15 (PR8 HA and PR8 NA), JV16 (PR8 HA and VN1203 NA), JV17 (PR8 HA and VN1203 NA with the stalk deleted), JV18 (PR8 HA and VN1203 NA with the PR8 stalk), JV22 (PR8 HA and VN1203 NA with the PR8 N terminus), JV19 (VN1203 HA and PR8 NA), JV20 (VN1203 HA and VN1203 NA), JV21 (VN1203 HA and VN1203 NA with the stalk deleted), JV23 (VN1203 HA and VN1203 NA with the PR8 stalk), and JV24 (VN1203 HA and VN1203 NA with the PR8 N terminus) were used.

FIG. 5.

Neuraminidase activity. The JV24 reassortant virus shows restored levels of NA activity. NA activity was measured at 12 h postinfection in MDCK cells and was normalized for HA in each strain at that time point. The mutant strains are compared to the JV20 virus, which contains both HA and NA from VN1203 and is set at 100% for NA activity. This graph shows the averages from three individual experiments.